Fungicide tolclofos-methyl [(O,O-dimethyl O-(2,6-dichloro-4-methylphenyl) phosphorothioate)], belonging to the group of organophosphorus compounds, has been usually applied for the effective control of soil born diseases by Rhizoctonia solani. However, its excessive usages have leaded to the accumulation of this compound on soil and ginseng itself. Therefore, we practiced the actual degradation of tolclofos-methyl using organophosphorus hydrolase (OPH). A gene encoding OPH from Flavobacterium sp. strain ATCC 27551 was cloned and expressed in Escherichia coli with or without signal peptide-encoding sequences under control of a T7 promoter. High-level expression of recombinant OPH was verified by specific OPH activity assay. In addition, although the extracellular secretion of OPH in E. coli has never been reported till now, secretion of the recombinant OPH was observed when a signal peptide of the gene was truncated. Recombinant E. coli strain removed a maximum of 80% of the organophosphorus compound tolclofos-methyl, as determined by an in vitro assay. The present study reports for the first time on the secretion of recombinant OPH not affected by signal peptide sequence in E. coli and biodegradation of tolclofos-methyl by extracellular secreted OPH from recombinant E. coli.